This invention relates to immunology and more particularly to a method of manufacture of immunogenic, compositions, to immunogens manufactured by the method, and to antibodies manufactured therefrom.
An immunogen is a molecule capable of eliciting an immune response in a vertebrate. The response elicited is believed to be determined by topographical shape characteristics of the immunogen. Immunogens are also called antigens i.e. ANTIbody GENerators because one aspect of the induced response involves the production of antibody molecules whose function is to lock onto the immunogen. Those areas of the immunogen to which the antibody molecule binds are variously referred to as the antigenic determinants, epitopes or haptens. The last term, namely hapten, is generally associated with the term carrier and this term refers to that part of the immunogen/antigen which interacts with cellular components of the vertebrate immune system.
These regions on the immunogen and the names used to define them should not be regarded as absolute. Thus the genus of vertebrates has immune systems which will recognize immunogens; but not all species necessarily recognize the same molecular areas as being haptenic areas or carrier areas. Within a species, the recognition of haptenic molecular areas can only be determined experimentally. Thus, mice will not necessarily process immunogens in the same way as would, for example, the immune system of Man. Furthermore, within a species, individual specimens will not respond to the same degree. This is because the immune response to an immunogen has a genetic (hereditary) component. Thus, some individuals will respond better to an immunogen while others may not respond at all.
The immune response to an immunogen is an integrated phenomenon in that a class of white blood cells called T lymphocytes, for example, reacts with the carrier determinants which in turn allows a class of white blood cells called B lymphocytes to transform and start producing antibodies to the antigenic determinants.
Each cell recognizes only one determinant and each antibody producing B cell (plasma cell) generates only antibody molecules of one given specificity. Hence, the immune system is said to be highly specific. Upon stimulation, these plasma cells multiply and thereby give rise to a clone of identical antibody secreting cells. If it were possible to isolate these identical antibody secreting cells, they would be referred to as monoclonal and the antibodies referred to as monoclonal antibodies.
FIG. 1 is a diagrammatic illustration of the response of a mouse to an immunogen/antigen. Under normal conditions each monoclonal antibody generated by the mouse in vivo mixes with other monoclonal antibodies so that a polyclonal antibody response eventuates.
Each antibody comprises a glycoprotein molecule. The portion of an antibody molecule embodying the characteristic of shape or molecular topography, or code sequence which enables it to bind and so for example neutralise the antigenic determinant or epitope of an antigen is known as a xe2x80x9cparatopexe2x80x9d. The paratope is conceptually a molecular region of a shape complementary to the epitope or to a part of the epitope of the antigen and is thought to reside in the so called hypervariable region of the antibody glycoprotein molecule.
Antibody producing lymphocytes are present in high concentration in the spleen but antigen reactive spleen lymphocytes cannot readily be cultured in isolation. However, monoclonal antibodies may be manufactured and isolated therefrom by use, for example, of techniques of hybridoma technology. In one such technique mice are first exposed to an antigen whereby the mouse develops antibodies. With reference to FIG. 2, spleen cells of the immunised mouse are fused with mouse myeloma cells. The growth of hybrid cells is promoted and the hybrids are screened for specific antibody secretion. Those useful are cultured or undergo further genetic stabilisation procedures. By this means specific monoclonal antibodies may be produced and isolated.
Selected antibodies, or mixtures thereof such as are produced in the method of FIG. 2, may be used to neutralise an antigen in an organism, a paratope of each antibody in effect forming a complex with an epitope of the antigen.
In anti-idiotypic immunology a second stage process shown in FIG. 3 is involved. Mouse 1 is first immunised with an antigen. Thereby giving rise to several clones of antibody producing cells. One cell line is chosen on the basis of the characteristics of the generated antibody and the antibody is referred to as Ab1. Ab1 is then used to immunise a second mousexe2x80x94mouse 2. The latter must have a genetic constitution very similar to, or identical with that of mouse 1. Mouse 2 generates monoclonal antibodies to Ab1, a subset of which may be directed against the paratope of Ab1. All the antibody subsets generated by mouse 2 against Ab1 may be referred to as Ab2 though the Ab2 subset specific for the paratope of subset 1 is sometimes referred to as Ab2 beta. The second mouse monoclonal antibody, Ab2, has an anti-paratope, that is to say having a molecular portion with a shape characteristic complementary to the paratope of the first antibody. If the epitope of the original antigen is considered to be xe2x80x9cmould positivexe2x80x9d, the paratope of the monoclonal antibody Ab1 can be considered to be a counterpart or xe2x80x9cmould negativexe2x80x9d and the paratope of the anti-Ab1 antibody that is the paratope of the Ab2 monoclonal antibody can be considered to replicate the xe2x80x9cmould positivexe2x80x9d. It will be understood however that in each case the replication is not exact. When used in a vaccine, the second monoclonal antibody, Ab2, functions as a harmless immunogen which stimulates production of AB3 antibodies in the vaccinated animal effectively producing immunity to the first antigen.
Anti-idiotypic vaccines are designed to be an interspecies approach so as to identify epitopes which induce neutralising antibodies in genetically diverse population. The approach requires that the anti-idiotypic vaccine candidates (Ab2) be inoculated into populations as diverse as sheep, chimpanzees and rabbits followed by antigen challenge to determine if the Ab3 carries the neutralising characteristics of the Ab1. If the challenge is successful Ab1 is further studied as a possible vaccine. If challenge is unsuccessful, the antibody is disregarded for its role as a vaccine. In this approach, the animal studies are meant to simulate the genetic diversity found in Man. The epitopes are therefore exclusively public in that they are capable of generating neutralizing responses in a number of different species.
According to one aspect the present invention consists of a method of treating an animal comprising the steps of:
(1) selecting from a pool of antibodies occurring in a first species of vertebrate a prototypic set the members of which are antibodies with specificity for a specific antigen or antigen epitope expressed in one or more members of the first species, and not expressed in members of a second species;
(2) utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of the second species, wherein said second species is selected such that the immune system of said second species does not recognise the specific antigen or antigen epitope uniquely expressed in one or more members of said first species, or said paratopic set or paratopic fragments thereof are utilized as an immunogen in an in vitro incubation system comprising cells derived from the same or a different species, to produce antibodies having a characteristic which is anti-paratopic with respect to said immunogen to produce a synthetic replicate of the specific antigen or epitope.
(3) introducing anti-paratopic antibodies produced in step 2 into the same or a different members of the first species selected in step 1.
According to a second aspect the present invention consists of a method of manufacture of an anti-paratopic antibody efficacious against mammalian infections comprising the steps of:
(1) selecting from a pool of antibodies occurring in a first species of vertebrate a prototypic set the members of which are antibodies with specificity for a specific antigen expressed in one or more members of said first species and not expressed in members of a second species;
(2) utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of the second species wherein said second species is selected such that the immune system of said second species does not recognise the specific antigen or antigen epitope uniquely expressed in one or more members of said first species, or said paratopic set or paratopic fragments thereof are utilized as an immunogen in an in vitro incubation system comprising cells derived from the same or a different species to produce one or more antibodies having a characteristic which is anti-paratopic with respect to the antigen or antigen epitope expressed in one or more members of said first species but not expressed in members of the second species; and
(3) selecting and purifying said anti-paratopic antibodies produced according to step 2.
In a preferred embodiment of the invention the anti-paratopic monoclonal antibodies are used to immunize a different member of the first species as that from which the prototypic set was selected.
For preference the pool of antibodies consists of naturally occurring human antibodies.
The prototypic set is a set of antibodies selected on the basis of effectiveness against a particular antigen, or epitope thereof, for example is a set of human antibodies selected from humans carrying antibodies resulting from exposure to HIV.
The antibodies, or paratypic paratope bearing segments of them, are utilized as an immunogen in a mouse host to produce mouse antibodies having anti-paratope characteristics.
The mouse antibodies are then screened first for their ability to specifically bind antigens or antigen epitopes in relation to the first species and then in respect of their effectiveness for inducing antibodies which bind the HIV in other members of the first species.
It will be understood that the response generated against the antigen or antigen epitope under consideration would be uncommon in nearly all species except the first species from which the prototypic set of antibodies was selected.